The influence of hygroscopic movement on seed dispersal in Daucus carota (Apiaceae)

Summary The influence of hygroscopic movement on seed dispersal in Daucus carota was examined. When relative humidity increases, umbels containing mature fruits close; when relative humidity drops, umbels open. Tests of the effectiveness of smalland large-angled umbels on dispersing seeds under various conditions demonstrate that umbels responding greatly to relative humidity (i.e. opening wide) lose seeds more quickly than do umbels responding little, and do not disperse them as far. As relative humidity increases, number of dispersing seeds drops to near zero. Response to changes in relative humidity within an umbel progressively increases from late August when dispersal begins. Umbels that slowly increase their response retain some seeds that may disperse over snow in winter. Individual variation in response to relative humidity is high among plants beginning dispersal at the same time. This variability is probably maintained by the variable consequences of dispersing seeds at different times.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Carrot (Daucus carota) hypocotyl transformation usingAgrobacterium tumefaciens

Summary Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, -glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.Abbreviations NPT II neomycin phosphotransferase II - Nos nopaline synthase - GUS -glucuronidase - CaMV cauliflower mosaic virus - 2,4-D 2,4 dichlorophenoxyacetic acid - PMSF phenylmethylsulfonyl fluoride     

Dynamics of Competition in Populations of Carrot (Daucus carota)

Populations of carrot (Daucus carota) were raised over a widerange of densities (79–5763 plants m^-2) to examine thedynamics of competition in terms of yield–density relationshipsand size variability, and to investigate the effects of nutrientsupply on competition. While the relationship between shootyield and density was asymptotic, the relationship between rootand total yield and density tended to be parabolic. For a giventime and density series the relationship between yield per unitarea and density could best be described by the model: y=w_mD_(1+aD)^b wherey is the yield per unit area,D is density,w_m, a andb arefitted parameters. The parametersw_m anda increased over timebut nutrient availability affected onlyw_m. An extension of thebasic yield-density model is proposed to describe the dynamicsof the yield–density relationship over time: y=kD_{[1+cexp(-rt)]{1+}     

The Effect of Dissolved Oxygen Concentration on Growth and Differentiation of Carrot (Daucus carota) Tissue

For suspension cultures of carrot tissue there is a criticallevel of dissolved oxygen concentration. Below this level, dryweight increased linearly with time while cell numbers increasedexponentially, creating ‘meristematic-like’ conditionsin the culture. These conditions favoured differentiation intoplantlets by embryogenesis while conditions above the criticallevel, when both cell dry weight and cell numbers increasedexponentially, favoured plantlet differentiation by rhizogenesis.Different light treatments affected the yield of structuresformed but did not prevent oxygen from controlling the differentiationpathway.     

Failure to establish a functional shoot meristem may be a cause of conversion failure in somatic embryos of Daucus carota (Apiaceae)

A carrot cell culture line was shown to be highly embryogenic, but plantlet recovery (conversion) was low (about 14%). The majority of somatic embryos that did not convert showed pronounced vacuolation in the apical notch, leading to their inability to form primary leaves and thereby convert. Comparisons of developing meristems in the shoot apical notch of converting somatic and zygotic embryos revealed similarities in cytoplasmic density and meristem organization between the two populations. Abscisic acid (ABA) was shown to significantly increase conversion in somatic embryos of globular, torpedo, and preplantlet stages (62%, 62.5% and 40%, respectively). Somatic embryos that were treated with 50 μM ABA showed retention of the highly cytoplasmic cells in the apical notch. Histological analysis showed a resemblance between shoot apices of converting somatic embryos and ABA-treated somatic embryos. ABA may be an induction agent for meristematic organization, or perhaps may cause cells of the apical notch to extend competence for determination as meristematic cells.     

Oleosomes (Spherosomes) from Daucus carota suspension culture cells

Isolated oleosomes from Daucus carota L. cells are lipid droplets consisting mainly of triacylglycerols (>97%) and very little protein (1–2%). The boundary between the lipid phase and the cytosol, which is visible on electron micrographs, is not built up by a true phospholipid-containing unit or half unit membrane. Enzymatic activities of lipid metabolism were not found to be associated with oleosomes with the exception of very low (contaminating) acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) and relatively high acyl-CoA hydrolase (EC 3.1.2.2) activities. The triacylglycerols exhibited a half life time of about 70 h, which is below the generation time of the cells (80–90 h). The fatty acid pattern of triacylglycerols was very similar to that of polar cellular membrane lipids.     

Expression of rabies virus G protein in carrots (Daucus carota)

Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4–1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.     

Occurrence of petaloid stamens in wild carrot (Daucus carota) from Sweden

Sterile petal-like stamens, reported by others in North American wild carrot, are here reported and illustrated from a wild carrot accession from Sweden. Although the North American material has been utilized already in carrot breeding, a more fundamental study of stamen petaloidy in botanical research dealing with flower differentiation is urged.     

Transformation and regeneration of carrot (Daucus carota L.)

A protocol is presented for the efficient transformation of carrot (Daucus carota L. cv. Nantaise) by Agrobacterium tumefaciens. The binary vector contained the marker gene -glucuronidase (GUS), driven by the 35S promoter of cauliflower mosaic virus, and the nptII gene, which confers kanamycin resistance. Highest T-DNA transfer rates were obtained by co-cultivating bacteria with hypocotyl segments of dark-grown seedlings on solidified B5 medium containing naphthaleneacetic acid and 6-benzylaminopurine. After 2 days, bacterial growth was stopped with antibiotics. Two weeks later, the explants were placed on agar containing the kanamycin derivate geneticin; antibiotic-resistant calli developed during the following 4 weeks. Suspension cultures were obtained from resistant calli and plants regenerated via somatic embryogenesis in liquid culture. The majority of plants were phenotypically normal and, depending on the Agrobacterium strain used, harbored single or multiple copies of the T-DNA. About equal levels of GUS activity were found in different organs of young plants up to 6 weeks after embryogenesis. In leaves of older plants, GUS activity was markedly reduced, whereas the activities in phloem and xylem parenchyma cells of developing tap roots were still high and fairly uniform. Thus, the 35S promoter may be a useful tool to drive the expression of transgenes in developing carrot storage roots.     

Effect of brassinosteroid on cell division and enlargement in cultured carrot (Daucus carota L.) cells

When added at 3.10^–6 mM to auxin-starved suspension cultured carrot (Daucus carota L.) cells, the steroid hormone 24-epibrassinolide (BR) induces cell enlargement but not cell division.This is at variance from the effect of 2,4-D which, in the same experimental conditions, restores cell division without having any effect on cell enlargement.Furthermore, in the tested experimental conditions, the effect of BR is dominant over the effect of 2,4-D when the two hormones are simultaneously supplied to the auxin-starved culture.Abbreviations BR brassinosteroid, 24-epibrassinolide - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - GA₃ gibberellic acid     

The Origin and Development of Embryoids in Suspension Cultures of Carrot (Daucus carota)

In suspension cultures of carrot, embryoids are initiated ator near the surface of characteristic cellular aggregates (embryogenicclumps) and are released from these aggregates as free-floatingstructures capable of further development into plantlets. Embryoiddevelopment is promoted by transfer from a medium containing2,4-D to one from which it is omitted. The structure of theembryogenic clumps, in these two media, has been studied bythe thin section technique involving fixation in glutaraldehydeand embedding in glycol methacrylate. The superficial cellsof the clumps are clearly marked off from the central cellsand contain large starch grains, a large central nucleus, abundantcytoplasm and a number of vacuoles. It is from individual superficialcells that the embryoids are seen to arise in the 2,4-D omittedmedium but it has not been possible to distinguish, in advanceof embryogeny, such cells from within the superficial cell layers.The fragmentation of the proliferating clumps in 2,4-D containingmedium and the release of embryoids into the 2,4-D omitted mediumis promoted by some of the superficial cells becoming free ofstarch and undergoing pronounced enlargement. A sequence of segmentations leading from single superficialcells of the clumps to globular embryoids has been traced. Thisdiffers from that reported to occur during early embryogenyfrom the egg cell of carrot. The resemblance to zygote embryogenyis closer from the late globular stage of embryoid development;embryoids then differ from zygotic embryos in their shortersuspensors. Attention is drawn to the initiation of additionalembryoids from the epidermis of some of the embryoids developingin culture.     

A comparison of methods for evaluating seed quality in carrots (Daucus carota)

Comparisons of embryo length, ISTA (International Seed Testing Association), cold (10 ^oC), controlled-deterioration, and slope tests for estimating seedling size variability and percentage seedling emergence were made using eighteen stocks of carrot seeds. The coefficient of variation (C.V.) of seedling weight was closely correlated with the C.V. of embryo length (r = 0–87 D.F., 16) and also with the C.V. of root length from the slope test (r = 0–63 D.F., 16). Poorer estimates of percentage seedling emergence were obtained from the cold test in all three sowings (correlation coefficients were between 0–65 and 0–70 D.F., 16) compared with the ISTA, controlled deterioration, and slope tests. For these three tests the correlation coefficients varied between 0–80 and 0–87 (D.F., 16) for all sowings. The embryo test is a rapid (1 man hour per seed lot) and accurate method of assessing potential variability between seed lots. The slope test could provide a less labour demanding method of assessing potential variability than the embryo length test though the results would not be available immediately. However, the slope test could also provide as good an estimate of seedling emergence in the field as the ISTA or controlled-deterioration tests.     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

The effects of ripening and drying of carrot seed (Daucus carota) crops on germination

Carrot seeds taken from the parent plant were capable of germinating before the stage when maximum seed dry weight was reached but even after this stage, when the seed moisture content had fallen below 20%, improvement in seed germination characteristics continued. In the latter stages of seed growth losses by shedding were 12–20 kg/ha/day. For low density crops (10 plants/m²) the yield of viable seed was at a maximum in crops whose seed was harvested with a moisture content of between 20 and 40% but no consistent relationship could be established for high density crops (80 plants/m²). There were no effects of umbel order or plant density on mean germination time or spread of germination. At any early harvest, percentage germination was highest for primary-umbel-seeds and seeds from low density crops but the differences between the seed origins diminished with later harvests. Drying the seeds on the umbels improved the percentage germination, reduced the mean germination time and the spread of germination particularly at the early harvests compared with seeds removed from the umbels and germinated immediately without drying.     

Field experiments on seed dispersal by wind in ten umbelliferous species (Apiaceae)

This report presents data from experiments on seed dispersal by wind for ten species of the family Apiaceae. Seed shadows were obtained in the field under natural conditions, using wind speeds between four and ten m/s. The flight of individual seeds was followed by eye, and seed shadows were acquired, with median distances varying from 0.7 to 3.1 m between species. Multiple regression models of wind speed and seed weight on dispersal distance were significant for six out of ten species; wind speed had significant effects in seven cases, but seed weight only once. A good correlation between mean terminal falling velocity of the seeds of a species and median dispersal distance, indicates the promising explanatory power that individual terminal velocity data might have on dispersal distance, together with wind speed and turbulence. The theory that seeds that seem to be adapted to wind dispersal travel much longer distances than seeds that have no adaptation was tested. Flattened and winged seeds were indeed found to be transported further by wind, but not much further. Moreover, the species with wind-adapted seeds were also taller, being an alternative explanation since their seeds experienced higher wind speeds at these greater heights. Furthermore, flattened and winged seeds were disseminated from ripe umbels at lower wind speeds in the laboratory. This means that the observed difference in dispersal distance would have been smaller when species specific thresholds for wind speed were incorporated in the field experiments. We argue therefore, that seed morphology is not always the best predictor in classifying species in groups with distinctly different dispersal ability.     

Mitochondrial DNA diversity and male sterility in natural populations of Daucus carota ssp carota

Mitochondrial variability was investigated in natural populations of wild carrot (Daucus carota ssp carota) in different regions: South of France, Greece, and various sites in the Mediterranean Basin and Asia. Total DNA was digested with two restriction endonucleases (EcoRV and HindIII) and probed with three mitochondrial DMA-specific genes (coxI, atp6, and coxII). Twenty-five different mitochondrial types were found in 80 analyzed individuals. Thirteen mitotypes were found among the 7 French populations studied. On average, 4.4 different mitotypes were observed per population, and these mitotypes were well-distributed among the populations. All of the mitochondrial types were specific to a single region. However, the proportion of shared restriction fragments between 2 mitotypes from different regions was not particularly lower than that which occurred among mitotypes from a single region. On the basis of the sexual phenotype [male-sterile (MS) or hermaphrodite] of the plants studied in situ and that of their progeny, 2 mitotypes were found to be highly associated with male sterility. Eighty percent of the plants bearing these mitotypes were MS in situ, and all of these plants produced more than 30% MS plants in their progeny. This association with male sterility was consistent in several populations, suggesting an association with a cytoplasmic male-sterility system. Moreover, these two mitotypes had very similar mitochondrial DNA restriction patterns and were well-differentiated from the other mitotypes observed in wild plants and also from those observed in the two CMS types already known in the cultivated carrot. This suggests that they correspond to a third cytoplasmic sterility.     

Methotrexate and aminopterin effects on growth and regeneration in Daucus carota

Daucus carota L., callus was cultured on various levels of the folate analogs, methotrexate (4-amino-10-methylfolic acid, amethopterin) and aminopter in (4-aminofolic acid). Callus growth was inhibited as analog concentrations were increased from 0.01 M to 10 M. Methotrexate concentrations in excess of 10 M were lethal. In contrast, concentrations of aminopterin in the range of 10 to 100 M resulted in renewed growth and somatic embryogenesis leading to plant regeneration. This plant regeneration occurred even in the presence of 5.0 mg/l 2,4-D or NAA (concentrations up to fifty times higher than that required to maintain callus growth). These observations reveal that aminopterin at high concentrations, but not methotrexate, triggers somatic embryogenesis in the presence of auxin. All tested levels of aminopterin permitted regeneration in the absence of auxin.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - DHFR EC 1.5.1.3, dihydrofolate reductase or 5,6,7,8,- tetrahydrofolate: NADP⁺ oxidoreductase - C1 single carbon - dTMP deoxyribothymidine-5 — monophosphate Technical Article No. 21590 from Texas Agricultural Experiment Station     

Freeze Preservation of Somatic Embryos and Clonal Plantlets of Carrot (Daucus carota L)

Cell suspensions of carrot (Daucus carota L.) can be cryopreserved by slow freezing (about 2 C per minute) in medium containing dimethylsulfoxide as a cryoprotectant. After storage in liquid nitrogen and thawing they demonstrate a high viability and are able to resume growth. Such a method entirely fails to preserve clonal plantlets; somatic embryos cease organized development at the time of freezing and recover growth only by secondary embryogenesis. Modification of the procedure, involving the removal of superficial moisture from cryoprotectant-treated embryos and plantlets and enclosing them in a foil envelope before freezing, greatly improves their survival potential. The use of dimethylsulfoxide at levels between 2.5 and 20% (v/v) and freezing at rates between 1 and 5 C per minute yielded viable preparations under appropriate thawing conditions. In general, treatments which increased tissue dehydration before or during freezing were most successful when followed by relatively slow thawing. Conversely where dehydration to a lesser degree was achieved, more rapid thawing was advantageous. Postthawing washing or inoculation into liquid media was inhibitory to recovery. On semisolid regrowth medium, somatic embryos resumed normal development, whereas in plantlets the root and shoot meristem regions gave rise to new growth. In both cases, inclusion of activated charcoal in the medium promoted organized growth.